Abstract Aging and carrying the APOE e4 allele are among the strongest risk factors for developing late-onset Alzheimer?s disease (AD). Several lines of evidence reveal an increase in endosomal trafficking proteins in the brains of APOE e4 allele carriers decades before the onset of cognitive decline. However, there is a major gap in our understanding of the critical mechanisms by which the ApoE4 protein regulates endosomal trafficking. The activity of the ATP binding cassette 1 (ABCA1) has an important role in the endocytosis of lipid-poor ApoE to initiate its endosomal recycling. This facilitates lipidation of ApoE and its recycling into the extracellular space. Loss of ABCA1 activity increases lipid-poor and aggregated ApoE particles. In humans, genetic loss-of-function mutations in ABCA1 are associated with increased AD risk. We hypothesize that aggregation of lipid-poor ApoE4 is at the root of endosomal trafficking dysregulation, and that activation of ABCA1 to lipidate ApoE decreases its aggregation and favors its endosomal recycling. To address this hypothesis, we propose the following three Specific Aims. In Aim 1, we determine the mechanisms of how ApoE and ABCA1 activity regulate endosomal trafficking in primary astrocytes, neurons and microglia. In Aim 2, we determine the effect of aging, APOE4 genotype and enhancing ABCA1 activity on ApoE aggregation and endosomal trafficking pathways in brains of ApoE targeted replacement mice, and in existing well-characterized human brain tissues that differ by APOE genotype and cognitive state. In aim 3, we propose to develop an 18-F CS-6253 PET imaging modality to assess the effect of APOE e4 and aging on ABCA1 brain activity in vivo. Achieving our aims will provide a detailed understanding of the effect of APOE4 on endosomal trafficking proteins, demonstrating a novel concept that activation of ABCA1 can ameliorate the congestion of ApoE4 containing endosomes. The information obtained is of major significance to understanding early events that predispose to AD pathology and developing therapeutic strategies focused on enhancing ABCA1 activity.